Title: CDK2 inhibitors for protecting hearing loss PROJECT SUMMARY Hearing loss is a major health concern in our society, affecting over 360 million people worldwide (World Health Organization, 2017). Cisplatin chemotherapy causes permanent hearing loss in 40-60% of treated cancer patients. To date, no drugs have been approved by the Food and Drug Administration (FDA) for protection from cisplatin-, noise-, or age-related hearing loss. Most candidate compounds currently in pre-clinical trials are related to antioxidants, vitamins, and glutathione metabolism, and thus many of these compounds, such as sodium thiosulfate, can interfere with cisplatin?s ability to kill the tumor cells. We recently conducted unbiased high-throughput screens of bioactive compounds (total of 4,385 unique compounds) in a cochlear ear cell line and identified cyclin dependent kinase-2 (CDK2) as an important therapeutic target for cisplatin-induced cell death and hearing loss. In the following focused screen of an additional 187 CDK2 inhibitors that have desirable drug-like properties, we identified AZD5438 as the top hit, exhibiting an IC50 of 540 nM in the cochlear cell line and an excellent IC50 of 5 nM ex vivo in mouse P3 cochlear explants treated with cisplatin. AZD5438 was the most potent CDK2 inhibitor tested in our cochlear explant studies. Furthermore, by local delivery of AZD5438 to adult FVB mice, the compound showed full protection against cisplatin-induced ototoxicity as measured by Auditory Brainstem Response (ABR) thresholds and cochlear histology. AZD5438 also protected against cisplatin induced hair cell loss in vivo in zebrafish lateral line neuromasts at 100 nM. Here we will evaluate the potential to repurpose the anti-cancer small molecule AZD5438, an orally bioavailable CDK2 inhibitor that has already been found to be tolerated in healthy male volunteers and solid-tumor patients in phase I and phase II clinical trials, for protection against cisplatin-induced hearing loss. Our Specific Aim is to test whether AZD5438 protects from cisplatin-induced hearing loss by systemic delivery in a mouse model. Our approach is to administer AZD5438 by oral gavage to adult FVB mice treated with cisplatin, measure their ABR thresholds and analyze their cochlear histology. The maximum non-toxic dose of oral AZD5438 will be experimentally determined and tested for hearing-protective effects. This work will shed light on the possibility of using AZD5438 in an oral formulation to combat cisplatin-induced hearing loss. In comparison to local delivery, oral delivery of an effective pharmaceutical product has the advantage of patient convenience. In the future, we will also test the efficacy of oral AZD5438 to protect from noise- and age- related hearing loss. If oral delivery of AZD5438 proves protective in this study, we will apply for IND-enabling SBIR phase II studies for cisplatin- induced hearing loss in cancer patients. As the main inventors for CDK2 inhibitors for hearing loss, both founders of Ting Therapeutics LLC have already obtained the exclusive patent rights for AZD5438 and filed patent applications in Europe, China, Japan and Hong Kong, and are negotiating for licensing the US patent rights. Oral delivery of AZD5438, if successful, has the potential to be a significant step forward in treating cancer patients against cisplatin-induced hearing loss.